The present invention relates to a method for selective determination of the .alpha.-ketoisocaproate content in samples of body fluids, especially serum samples, by enzymatic reaction in the presence of coenzyme, and to an analytical kit for such a determination.
Ketoisocaproate (KIC) is a metabolite which is generated, normally as an intermediate, in the catabolism of leucine to acetyl CoA and acetoacetate. The relevant portion of the pathway in question is shown below, along with the corresponding portions of the catabolic pathways for two other branched-chain amino acids, isoleucine and valine: ##STR1##
A hereditary anomaly in neonates, called maple syrup urine disease (leucinosis) because of the characteristic odor of the urine, results in a blockage in the breakdown of KIC, as well as of the two other branched-chain keto acids formed, respectively, from valine and isoleucine, due to a failure of normal oxidative decarboxylation. The genetic defect thus affects the branched-chain keto-acid dehydrogenase, resulting in the accumulation of KIC and the other branched-chain keto acids in the serum and, subsequently, in the urine. KIC has a toxic effect on the brain and can, when present in high concentration, result in death of children in infancy or in impaired brain development.
The only successful therapy known for maple syrup urine disease involves adherence to a special diet, in which the intake of leucine, valine and isoleucine in the food is distinctly restricted compared with normal. Frequent checks of the plasma leucine or KIC levels are necessary to monitor the therapy. A peculiarity of this disease is that when there are febrile infections, as occur very often in infancy, there is likewise an increase in leucine and KIC in the blood, which is frequently dangerous. The essential prerequisite for effective therapy is, therefore, a good method of amino acid analysis or KIC analysis. Particularly suitable for bedside monitoring of the patient's metabolism is determination of KIC in urine. A good KIC analysis is an absolute prerequisite for this.
In other metabolic abnormalities and, particularly, in the context of diabetes and liver damage, there may likewise be a dangerous accumulation of physiologically unacceptable levels of KIC, so that selective determination thereof is of considerable importance.
The problem associated with KIC analysis is that both the serum and the urine also contain relatively high concentrations of the two other branched-chain keto acids (ketomethylvalerate=KMV; ketoisovalerate=KIV), which are chemically very similar to KIC and interfere with the KIC determination. Accordingly, there is an extensive literature (twenty-eight publications over the last ten years) dealing with chromatographic separation of these keto acids, after appropriate derivatization, by gas chromatography or high performance liquid chromatography (HPLC). See, for example, J. Chromat. 400 (1987) 91-99. These chromatographic methods are sensitive and reliable but require elaborate apparatus and are rather time-consuming. For this reason, KIC analyses have heretofore been carried out only in specially equipped analytical laboratories.
Although it is known that branched-chain 2-keto carboxylic acids can be analyzed reliably, rapidly and with little effort by enzymatic conversion, to date only the total concentration of the three said keto acids has been measured. See G. Livesey and P. Lund in Biochem. J. 188 (1980) 705-713, particularly at page 711; METHODS IN ENZYMATIC ANALYSIS (Bergmeyer, H. K., ed.) Vol. VIII pages 318-329, especially page 324, Verlag Chemie, Weihneim 1985; and Anal. Biochem. 162 (1987) 536-539.